CN116376920A

CN116376920A - Preparation method and application of CEACAM5 targeted CAR-T cells

Info

Publication number: CN116376920A
Application number: CN202310226162.6A
Authority: CN
Inventors: 李丹; 肖义泰; 梅超明; 杨帆; 单鸿
Original assignee: Fifth Affiliated Hospital of Sun Yat Sen University
Current assignee: Fifth Affiliated Hospital of Sun Yat Sen University
Priority date: 2023-03-10
Filing date: 2023-03-10
Publication date: 2023-07-04

Abstract

The invention provides a preparation method and application of CEACAM5 targeted CAR-T cells. In particular, the invention provides a Chimeric Antigen Receptor (CAR) whose antigen binding domain comprises a CEACAM 5-targeted nanobody sequence. The invention also provides a CAR plasmid, a viral vector and a CAR-T cell containing the CAR, wherein the CAR plasmid, the viral vector and the CAR-T cell can express a chimeric antigen receptor of targeted CEACAM 5. The CAR-T cells of the invention can be used for the treatment of CEACAM5 positive tumors.

Description

Preparation method and application of CEACAM5 targeted CAR-T cells

Technical Field

The invention relates to the field of biotechnology and immunotherapy, in particular to CEACAM 5-targeted CAR-T cells, and preparation and application thereof.

Background

Colorectal cancer (Colorectal cancer, CRC) is one of the most common malignancies, one of the leading causes of cancer death worldwide. Approximately 25% of patients are in stage iv at the first visit, and 25-50% continue to develop metastatic CRC, although presenting with early disease. The prognosis for metastatic CRC remains poor, with bit survival rates of only 18.5% in the 5 years of the United states and Europe. Standard conventional treatments for CRC are surgery, chemotherapy and radiation. Chimeric antigen receptor T (Chimeric antigen receptor T-cell, CAR-T) cells are one of the new options for CRC treatment. CAR-T cells consist of variable fragments of antibodies specific for the antigen of interest, fused to T cells, and the modified ex vivo CAR-T cells are reinjected into the patient and recognized with the target antigen, effecting active transport to the tumor site, and further in vivo expansion and long-term persistence, recognizing in a major histocompatibility complex-independent manner the activation of signaling pathways inside T cells by co-stimulatory domains such as CD28 or 4-1BB, and promoting cytotoxicity and tumor apoptosis by release of cytokines such as granzyme B, perforin, IL-2, IFN- γ, and the like.

CAR-T cell therapies for solid tumors still face many challenges. One of the important problems is the lack of a suitable CAR-T cell target antigen in solid tumors, which in most humans is also present in healthy tissue, resulting in non-targeted toxicity. Carcinoembryonic antigen cell adhesion molecule 5 (Carcinoembryonic antigen cell adhesion molecule, CEACAM 5), also known as CD66e or carcinoembryonic antigen (CEA), is a hyperglycosylated membrane-bound glycoprotein belonging to the CEA family. CEACAM5 has long been considered an attractive colorectal cancer target because it is highly expressed in the form of high antigen density almost exclusively in colorectal tumors, but limited expression in normal tissues. Studies have shown that CEACAM5 is expressed in more than 80% of colorectal cancers. This makes CEACAM5 a promising target for new therapies for CRC. Therefore, the development of the CEACAM 5-targeted CAR-T cells has a great application prospect for improving the immunotherapy effect of patients with advanced CRC.

CAR is composed of four components, an extracellular region, a hinge region, a transmembrane region, and an intracellular signaling region. The extracellular domain has flexible splicing functions and determines antigen specificity, and is a functional component for recognizing tumor antigens. The targeting domain of a CAR is based mainly on Fab or single chain variable fragments, typically monoclonal antibodies, which are widely used due to their compact size, high affinity and specificity. Nanobodies have unique potential in developing various forms of CAR-T compared to traditional single chain antibody fragments. In addition to its natural high binding capacity, nanobodies also have more advantageous structures in terms of immunogenicity, solubility and stability in vivo. Nanobodies do not render the body immunogenic due to the lack of linkers that result in the body being immunogenic. In general, nanobodies require only minor sequence modifications to the humanization process. In addition, single chain antibody fragments can lead to CAR-T failure induced by spontaneous activation, which is mainly related to the framework regions that make up the single chain antibody fragment portion of the CAR. This is one of the important reasons for the failure of CAR-T to treat solid tumors. In addition, nanobodies avoid potential disruption of interactions between the variable and constant regions and exposure to hydrophobic patches. Such disrupted interactions and hydrophobic residues may severely affect solubility and stability. Based on these advantages, nanobodies are expected to be potential new choices in CAR-T design.

Based on the background above, we developed CAR-T cells targeting the CRC specific target CEACAM5 using the sequence of CEACAM5 targeting nanobody and evaluated their antitumor activity at the cell level and in mouse tumor models.

Disclosure of Invention

The invention provides preparation and application of a CAR-T cell targeting CEACAM5 positive cells, wherein the CAR-T cell can express a chimeric antigen receptor targeting CEACAM5, specifically identify and kill CEACAM5 high-expression tumor cells, and is suitable for treating CRC.

Specifically, the content of the invention comprises:

in a first aspect, the invention provides a gene sequence of a CEACAM 5-targeted nanobody, which has a nucleotide sequence shown in SEQ ID NO. 2.

In a second aspect, the invention also provides a CAR plasmid comprising the gene sequence of the CEACAM5 targeting nanobody of the first aspect (referred to as Nb 41) capable of expressing a CEACAM5 targeting chimeric antigen receptor comprising a CEACAM5 targeting signal peptide, an antigen binding domain, a transmembrane domain, an intracellular co-stimulatory domain, and an intracellular domain.

Further, the signal peptide is selected from the domain of CD8 leader, and has a nucleotide sequence shown in SEQ ID NO. 1.

Further, the transmembrane domain is selected from a CD8 linker domain and a CD8 TM domain, and the nucleotide sequence of the transmembrane domain is shown as SEQ ID NO. 3.

Further, the intracellular co-stimulatory domain is selected from the group consisting of the intracellular co-stimulatory domains of 4-1BB, and has a nucleotide sequence as shown in SEQ ID NO. 4.

Further, the intracellular domain is selected from the intracellular domain of CD3 zeta, and has a nucleotide sequence shown in SEQ ID NO. 5.

In a third aspect, the invention also provides a CAR-T lentivirus comprising a CAR vector according to the second aspect.

In a fourth aspect, the present invention also provides a CAR-T cell expressing the CAR gene according to the first aspect, wherein the CAR-T cell is designed according to the specific interaction between the antigen binding domain of CEACAM5 and CEACAM5, and is capable of targeting and killing tumor cells highly expressed by CEACAM5, and is used for immunotherapy of CRC, improving the therapeutic effect, and significantly prolonging survival time of mice after CRT cell therapy (day 24 is observed).

In a fifth aspect, the invention also provides a method of treating colorectal cancer comprising administering to a subject in need thereof an appropriate amount of a plasmid, virus, or cell according to the second, third, or fourth aspects of the invention.

TABLE 1

Domain	Sequence(s)
		CD8 leader	ATGGCCCTGCCCGTGACCGCCCTGCTGCTGCCCCTGGCCCTGCTGCTGCACGCCGCCAGACCC
Nb41	CAGTTGCAGCTCGTGGAGTCTGGTGGAGGCTTGGTGCAGGCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGAAGCCTCTTCAG GATCAATGCCATGGCCTGGTTCCGCCAGGCTCCAGGGAAGCAGCGCGAGTTGGTCGCAGCTATTACTAGTGCTGGTAGTACAAACTATG CAGATTTCGTGAAGGGCCGATTCACCATCTCCGCAGACAACGCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAAACCTGAGGAC ACAGCCGTCTATTACTGTAATACACCCTGGCCCGTAGGGAGGGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAGAACCCAA GACACCAAAACCACAACCA
		CD8 linker & TM	ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGC GGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTC TCCTGTCACTGGTTATCACCCTTTACTGC
4-1BB	AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTG CCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAA
		CD3ζ	AGAGTGAAGTTCAGCAGAAGCGCCGACGCCCCCGCCTACCAGCAGGGCCAGAACCAGCTGTACAACGAGCTGAACCTGGGCAGAAGAGA GGAGTACGACGTGCTGGACAAGAGAAGAGGCAGAGACCCCGAGATGGGCGGCAAGCCCCAGAGAAGAAAGAACCCCCAGGAGGGCCTGT ACAACGAGCTGCAGAAGGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAGAGAAGAAGAGGCAAGGGCCACGACGGC CTGTACCAGGGCCTGAGCACCGCCACCAAGGACACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCCAGA

Drawings

FIG. 1 shows construction of a CAR-T cell expression plasmid according to the present invention. Wherein a is a schematic representation of a CAR-T cell expression sequence; b is linearization vector after PCR detection of expression fragment and vector fragment digested with high fidelity enzyme.

FIG. 2 is a schematic representation of the preparation and identification of CAR-T cells according to the present invention. Wherein A is fluorescence and white light visual field pictures of the transfected CAR-T cells; b is the ratio of CAR-T cell positive cells after transfection as detected by flow cytometry.

FIG. 3 is an experiment of the cell function of CAR-T cells according to the present invention. Wherein A and B are B41-CAR-T cells and cells after Co-culture of Mock-CAR-T with LS174-T and HT-29A killing effect map; c and D are cytokine secretion after co-culture of different tumor cells with B41-CAR-T cells and Mock-CAR-T cells, representingP < 0.0001。

FIG. 4 is an in vivo tumor suppression experiment of CAR-T cells according to the present invention. Wherein a is a picture of tumor bioluminescence imaging of a CAR-T cell treated subcutaneous tumor mouse at different time points; b is fluorescence intensity quantification of bioluminescence imaging of subcutaneous tumors at different time points; c is a tumor volume change curve obtained by measuring subcutaneous tumors at different time points; d is the survival curve of mice after treatment of tumors in the different groups.

Description of the embodiments

The following examples are intended to be illustrative of the present invention, but not limiting. Unless otherwise indicated, the examples were all under conventional experimental conditions.

Human colorectal cancer cell line (HT-29, LS174-T) and human T-lymphoma cell (Jurkat) human embryonic kidney cell 293 (HEK 293) cells were purchased from ATCC. Female Balb/c nude mice of 4 weeks old were purchased from Guangzhou City, chemie Biotech Co. Lentiviral packaging plasmids (pCDH-CMV, psPAX2, pMD2.G plasmids) were purchased from Addgene, inc. of America. Restriction endonucleases (BamHI, xbaI) were purchased from New England Biolabs. Clone-related reagents (Phanta_Max Super-Fidelity DNA Polymerase, clonExpress_MultiS recombinant cloning kit, gel recovery kit, plasmid miniprep kit, etc.) were purchased from Nanjinouzan Biotechnology Co. ELISA kits (IFN-. Gamma., IL 2) were purchased from Absin company. Lipofectamine ™ 3000 transfection reagent was purchased from Invitrogen corporation. Opti-MEM reduced serum medium was purchased from Thermo Fisher company. The enhanced CCK-8 kit was purchased from Biyun Tian Biotechnology Co. D-Luciferin potassium fluorescein salt was purchased from Perkinelmer. Luciferase reporter lentiviruses were customized by igeebio.

EXAMPLE 1 construction of Gene expression vectors

Firstly, synthesizing a gene fragment according to a gene sequence, taking the synthesized fragment as a template, inputting an insert fragment and a pCDH-CMV vector sequence by using primer Design software CE Design to generate a primer, checking, sending the primer to a company to synthesize the insert gene fragment by using a PCR reaction, separating a reaction product by gel electrophoresis, and cutting a target strip into gel to recover the gene fragment. The gene fragment vector is selected from Xbal and BamHI restriction endonuclease to linearize the circular vector, the reaction product is separated by gel electrophoresis, and the target band is cut into gel to recover the gene fragment. And (3) preparing a recombination reaction system to recombine target gene fragments into a linearization vector, converting the recombination products into DH5 alpha competent cells, coating the DH5 alpha competent cells onto a flat plate, culturing overnight, selecting 5 clones of the DH to expand and culture, and then sending a sequencing identification recombination result to obtain a vector with a correct sequencing result, namely the pCDH-Nb41-CD8 alpha-4-1 BB-CD3 zeta (B41 CAR) vector. The control vector (Mock-CAR) was designed to be identical to the pCDH-Nb41-CD8 a-4-1 BB-CD3 zeta vector except that it did not contain the nanobody Nb41 sequence.

We selected pCDH-CMV as the vector for lentiviral construction. The pCDH-CMV vector was cut into linearized vector by BamHI and XbaI double digestion, the molecular weight was 6232 bp, and the size of the molecular weight of agarose gel electrophoresis of the linearized vector after digestion was expected (FIG. 1B, lane 6-8). The B41-CAR sequence consisted of a CD8 signal peptide, nb41 nanobody sequence, transmembrane region and 4-1BB costimulatory region, CD3 zeta signal region (fig. 1A). Nb41 is a nano antibody sequence targeting CEACAM5 and is used for recognizing CEACAM5 antigen on the surface of tumor cells. As a control, a Mock-CAR without Nb41 targeting sequence was designed (fig. 1A). PCR amplified products by template all met the expectations (B41-CAR 1900 bp, mock-CAR was recombined from two fragments, 850 bp and 680 bp, respectively, FIG. 1B). And (3) carrying out enzyme-linked recombination on the linearization vector and the target gene fragment to obtain partial positive transformation clone, and continuing to amplify and extract plasmids after sequencing correctly to obtain the CAR-T expression plasmid.

EXAMPLE 2 construction of CAR-T cells

HEK-293 cells were recovered and subcultured and inoculated into 10 cm dishes until the cell density was observed at about 50% for transfection. DNA dilutions (solution A) were prepared in a ratio of 7.5. Mu.g of psPAX plasmid, 2.5. Mu.g of pMD2.G plasmid, 7.5. Mu. g B41-CAR or Mock-CAR plasmid to 375. Mu.L of opti-MEM and 15. Mu. L P3000.3000, lipo3000 dilutions (solution B) were prepared in 375. Mu.L of opti-MEM and 15. Mu.L of lipo3000, and the solutions A and B were gently mixed and allowed to stand, and then slowly added dropwise to the opti-MEM medium of HEK-293 cells. After transfection, 6 h was changed to fresh culture medium, and the first virus supernatant was collected at 48 h and stored at 4 ℃.72 h, collecting the virus supernatant of the second time, and storing at 4 ℃. The two virus supernatants were then mixed, centrifuged at 3000 g for 10 min to remove cell debris, 0.45 mm filter sterilized virus solution was added to the Jurkat cells after centrifugation, 24 h cell culture medium was changed after transfection of Jurkat cells, and after 72 h green fluorescence was observed under a fluorescence microscope. The cells that expressed positively were screened with 3. Mu.g/mL puromycin, and after 1-2 weeks of resistance screening, the surviving cells and cells that expressed positively were continued to be cultured with medium containing 1.5. Mu.g/mL puromycin.

The HEK-293 cells are transfected with pxPAX2, pMD2.G and a target plasmid through liposome, virus liquid is harvested for infecting the target cells, and cell stable strains are obtained after screening. Jurkat cells are an immortal human leukemia T cell line and are widely used to detect T cell activation and signaling mechanisms. Here, we assessed CAR-T in vivo and in vitro function in human Jurkat T cells. As shown in fig. 2A, jurkat cells transfected and screened under a fluorescence microscope were able to observe significant eGFP green fluorescence, indicating successful construction of CAR-T cell stable strains.

Example 3 flow cytometry detection of expression efficiency of CAR in Jurkat cells

Jurkat CAR-T cells were collected, centrifuged at 1000 rpm for 3 min, the medium was discarded, the cells were washed with PBS, and phenol red in the medium was removed so as not to affect the results. Cells were resuspended in PBS and analyzed by multicolor flow cytometry, channels were selected for B525-FITC, and the results were analyzed by Cytexpert software.

The positive expression efficiency of CAR was detected by flow cytometry, as shown in fig. 2B, the CAR-T cell positive cell ratio reached 63.72 ±3.47%.

Example 4 cell killing experiments of Jurkat CAR-T cells

To verify the function of CAR-T cells we selected LS174-T and HT-29 intestinal cancer cells highly expressed by CEACAM5, respectively plated on 96-well plates with approximately 5000 cells per well, 3 sub-wells per group. Jurkat CAR-T cells and Jurkat cells transfected with empty plasmids were counted and added to 96-well plates at a ratio of effector to target of 1:1, 2:1, 4:1, 8:1, 16:1 for co-incubation with CAR-T cells. Adherent cells were washed 3 times, added with DMEM medium containing 10 μl CCK-8 and 90 μl, incubated at 37 ℃ for 2-4 h, absorbance at 450 nm was measured for each well using an enzyme-labeled instrument, and cell viability was normalized using the formula.

The results show that B41-CAR-T cells can specifically kill LS174-T and HT-29 tumor cells, and that this cell killing effect is positively correlated with the cell-effect target ratio (Effector to target ratio), whereas no significant cell death was observed after LS174-T and HT-29 cells were co-incubated with Mock-CAR-T cells (FIGS. 3A, B). These results indicate that B41-CAR-T cells have a strong killing efficiency against CEACAM5 positive tumor cell lines.

Example 5 ELISA detection of the content of CAR-T cell secreting cytokines

The CAR-T cells were co-cultured with target cells LS174-T and HT-29 in a ratio of 8:1 for 24 hours, the co-culture supernatant was collected and cytokine secretion during co-culture was detected by ELISA. Compared with Mock-CAR-T cells, the results show that the secretion of cytokines IFN-gamma and IL-2 with anti-tumor activity in B41-CAR-T cells is obviously increasedP <0.0001 (fig. 3c, d). This suggests that the recognition of target cells by B41-CAR-T cells is dependent on the expression of CEACAM5 on the cell surface, mediating cell killing by secretion of cytokines.

EXAMPLE 6 in vivo treatment efficacy study of CAR-T cells

To verify the in vivo therapeutic effect of CAR-T cells, LS174-T-luc cell stable strain was constructed by transfecting CEACAM5 positive LS174-T cells with a slow virus solution. Inoculation of right thigh root of mice with 5X 10 ⁶ LS174-T-luc cells after resistance selection establish LS174-T-luc subcutaneous tumor model. The tumor size was measured daily after one week with calipers and the tumor volume was calculated using the following equation: tumor volume= (width) ² X length/2. When the tumor volume grows to about 200-300 mm ³ Mice were randomly divided into 3 groups (n=6): a group of CAR-T cells,mock group, PBS group. CAR-T cells (1X 10) were injected by tail vein ⁷ ) Injection time points were day 0 and day 6, respectively. The body weight and tumor size changes of the mice were recorded and tumor size was monitored dynamically by bioluminescence imaging (BLI). Time points recorded are

days

0, 3, 6, 10, 13, 20, 26, 34. Mice were injected with D-potassium luciferin working solution and anesthetized with isoflurane, and then imaged to detect luciferase signals, BLI signals were analyzed by IVIS imaging system. Mice were euthanized on day 35. Meanwhile, in any of the following cases, animals were considered to reach the experimental end point and euthanized: tumor size exceeds 1.4. 1.4 cm ³ The method comprises the steps of carrying out a first treatment on the surface of the Ascites occurs; weight loss>20% of a base; or any painful physical sign.

Experimental results show that B41-CAR-T cells were able to significantly inhibit the growth of LS174-T-luc subcutaneous tumors (fig. 4A). The tumor fluorescence and volume change curves showed that B41-CAR-T cells were able to significantly inhibit tumor growth, and that there was no significant difference between Mock-CAR-T treated and PBS control treated groups (fig. 4B, c). Survival curves showed that B41-CAR-T cell treatment significantly prolonged survival of mice (day 24 was observed, fig. 4D). These results indicate that B41-CAR-T cells have a strong in vivo tumor suppression effect.

Claims

1. A nucleotide sequence encoding a CEACAM5 targeting nanobody, the domain of the nucleotide consisting of CD8 leader, nb41, CD8 linker & TM, 41 bb, CD3 ζ.

2. A CAR plasmid vector, wherein the CAR plasmid comprises the CEACAM5 targeting nanobody nucleotide sequence of claim 1.

3. A CAR-T viral vector comprising the CAR plasmid vector of claim 2.

4. A CAR-T cell expressing the CEACAM5 targeted nanobody of claim 1.

5. Use of a nucleotide sequence, a plasmid, a viral vector, a CAR-T cell according to any one of claims 1-4 for the preparation of a medicament for the treatment of a tumor, said tumor being CEACAM5 positive.